The release of neurotransmitters from presynaptic terminals is the final response of a nerve to the excitatory and inhibitory inputs that converge upon it. Neurotransmitter release at the presynaptic terminal of neurons is primarily initiated by the entry of calcium through voltage-gated calcium channels (Smith and Augustine, Trends Neurosci. 11:458-464, 1988; Robitaille et al., Neuron 5:773-779, 1990). Exocytosis of synaptic vesicles occurs at specialized regions of the nerve terminal called active zones. These zones may contain clusters of presynaptic calcium channels that supply calcium for neurotransmitter release (Pumplin et al., Proc. Natl. Acad. Sci. USA 78:7210-7214, 1981; Pumplin, J. Neurocytol. 12:317-323, 1983; Zucker, J. Physiol. 87:25-36, 1993). The entry of calcium through voltage-gated calcium channels couples electrical activity to secretion of synaptic vesicles. Synaptic transmission is initiated within 200 .mu.s after the arrival of the action potential at the synaptic terminal. The brief rise in Ca.sup.++ concentration to the level necessary for exocytosis likely occurs only in proximity to the calcium channels (Llinas et al., Biophys. J. 33:289-322; Cope and Mendell, J. Neurosci. 47:469-478, 1982).
A combination of electrophysiological and pharmacological criteria have defined four main types of high-voltage-activated calcium channels that are widely distributed in mammalian neurons. These are .omega.-conotoxin-GVIA-sensitive N-type calcium channels, .omega.-agatoxin IVA-sensitive and .omega.-conotoxin-MVIIC-sensitive P-type and Q-type calcium channels, and dihydropyridine-sensitive L-type calcium channels (for reviews see Bean, Annu. Rev. Physiol. 51:367-384, 1989; Hess, Ann. Rev. Neurosci. 13:337-356, 1990; Tsien et al., Trends Pharmac. Sci. 12:349-354, 1991; Miller, J. Biol. Chem. 267:1403-1406, 1992; Zhang et al., Neuropharmacology 32:1075-1088, 1993). Several lines of evidence indicate that N-type channels, at least in part, are responsible for the calcium influx that triggers transmitter release in many neurons. Antibodies against .omega.-conotoxin GVIA (.omega.-CTx GVIA) or fluorescent toxin derivatives label active zones on the terminals of motor neurons at the frog neuromuscular junction (Robitaille et al., Neuron 5:773-779, 1990; Cohen et al., J. Neurosci 1:1032-1039, 1991). Immunocytochemical studies with specific site-directed anti-peptide antibodies indicate that N-type channels are located along the length of dendrites and in synapses formed on the dendrites of many brain neurons (Westenbroek et al., Neuron 9:1099-1115, 1992). In contrast, antibodies to L-type channels recognize calcium channels in cell bodies and proximal dendrites, but give no detectable staining of presynaptic terminals in brain (Ahlijanian et al., Neuron 4:819-832, 1990). In addition, .omega.-CTx-GVIA inhibits transmitter release in a variety of mammalian neuronal preparations (Hirning et al., Science 239:57-60, 1988; Horne and Kemp, Br. J. Pharmacol. 103:1733-1739, 1991; Takahashi and Momiyama, Nature 366:156-158, 1993; Luebke et al., Neuron 11:895-902, 1993; Turner et al., Proc. Natl. Acad. Sci. USA 90:9518-9522, 1993; Wheeler et al., Science 264:107-111, 1994), thus supporting the hypothesis that N-type channels play a role in controlling neurotransmitter release in the central nervous system. Similarly, P-type and Q-type channels (collectively P/Q-type channels) have been implicated in neurotransmitter release in mammalian neurons. N-type channels appear to be the dominant form in presynaptic terminals of the peripheral nervous system, and P/Q-type channels in presynaptic terminals of the central nervous system.
Molecular cloning has identified the primary structures of the main pore-forming .alpha.1 subunit of five distinct classes of calcium channels (classes A, B, C, D, and E) found in rat brain. Cloned neuronal .alpha.1.sub.C and .alpha.1.sub.D subunits are components of L-type channels, while the .alpha.1.sub.B subunit is a component of N-type channels (Dubel et al., Proc. Natl. Acad. Sci. USA 89:5058-5062, 1992; Williams et al., Neuron 8:71-84, 1992a; Williams et al., Science 257:389-395, 1992b; Westenbroek et al., Neuron 9:1099-1115, 1992; Stea et al., Neuropharmacology 32:1103-1116, 1993). .alpha.1.sub.A encodes Q-type calcium channels and may also encode P-type calcium channels (Snutch and Reiner, Curr. Opin. Neurobiol. 2:247-253, 1992; Tsien etal., Trends Pharmac. Sci. 12:349-354, 1991; Mori et al., Nature 350:398-402, 1991; Sather et al., Neuron 11:291-303, 1993; Zhang et al., Neuropharmacology 32:1075-1088, 1993). The deduced amino acid sequence of .alpha.1.sub.B shares overall structural features with other calcium channel .alpha.1 subunits. It is composed of four predominantly hydrophobic homologous domains (I-IV) that are linked by intracellular hydrophilic loops of various lengths.
The traditional approach to blocking neurotransmitter release has been to use compounds that bind to the neuronal voltage-gated calcium channels in a manner such that calcium entry through the channels is blocked. One of the difficulties in such an approach is the lack of specificity. As noted above, voltage-activated calcium channels that are found at sites in the body other than at presynaptic terminals appear to share structural features responsible for the movement of calcium through the channels. Accordingly, compounds that interact with the pore portion of calcium channels to block calcium entry into presynaptic nerve terminals will also block calcium channels at other sites throughout the body. Therefore, the traditional compounds for blocking neurotransmitter release have undesired side effects due to the blockade of additional calcium channels.
Due to the limited success for previously suggested compounds for the inhibition of neurotransmitter release, there is a need in the art for methods and compositions to screen for new inhibitors with specificity for presynaptic voltage-gated calcium channels. The present invention fulfills this need, and further provides other related advantages.